pMAL-p5e載體基本信息
載體名稱(chēng): | pMAL-p5e |
---|---|
質(zhì)粒類(lèi)型: | 大腸桿菌表達(dá)載體 |
高拷貝/低拷貝: | -- |
啟動(dòng)子: | -- |
克隆方法: | 多克隆位點(diǎn),限制性內(nèi)切酶 |
載體大小: | 5755bp |
5' 測(cè)序引物及序列: | -- |
3' 測(cè)序引物及序列: | -- |
載體標(biāo)簽: | -- |
載體抗性: | Ampicillin |
篩選標(biāo)記: | -- |
備注: | -- |
產(chǎn)品目錄號(hào): | -- |
穩(wěn)定性: | -- |
組成型: | -- |
病毒/非病毒: | -- |
The vector pMAL-p5E is designed to produce maltose-binding protein (MBP) fusions, where the protein of interest can be cleaved from MBP with the specific protease Enterokinase (NEB #P8070).
MBP fusions made with this vector include an N-terminal signal sequence, so the fusion protein is directed to the periplasm. The MBP has been engineered for tigher binding to amylose resin.
A gene or open reading frame is inserted into a restriction site of the vector polylinker, in the same translational reading frame as the malE gene (encoding MBP). The fusion protein produced from the vector can be purified by amylose affinity chromatography. The sequence coding for the five amino acids Asp-Asp-Asp-Asp-Lys is present just upstream of the KpnI site. This allows the protein of interest to be cleaved from maltose-binding protein with enterokinase.
pMAL-p5E cut with KpnI followed by treatment with the Quick Blunting Kit (NEB #E1201) produces a blunt end at the lysine codon. This allows blunt-end cloning of an insert where the first three nucleotides code for the first amino acid of the protein of interest, and enterokinase cleavage of the fusion produces a protein with no vector-derived amino acids.